A requirement to detect, size and count individual particles within a particle population suspended in a transparent fluid is frequently encountered in parenteral and general pharmaceutical analysis. Typical populations of interest include aggregates, contaminants, bubbles, and other particles.
Regulatory bodies such as the US FDA apply standards for parenteral injectable and ophthalmic solutions which specify the maximum concentration of particles larger than certain sizes which the solution may contain. The medical reasons for specifying such maximum allowed concentrations is that particulates larger than a certain size, conventionally defined by their equivalent sphere diameter (ESD), can have adverse effects on the patient when injected or placed on the eyes. These standards state that the size of particulates will be measured by a light obscuration instrument or, if the fluid is not suitable for such an instrument, by filtration followed by visual microscopy.
The light obscuration technique consists in passing the particles, one at a time, through an optical beam which then impinges on an optical detector. A threshold is applied to distinguish signals resulting from particles from noise variations. The particle size is determined by comparing, via a calibration table, the reduction in detector signal for each particle with the reduction when polystyrene (PS) spheres of known size are passed through the beam. The system must be recalibrated by the user at regular intervals.
The disadvantages of the light obscuration technique stem from the fact that particles in intravenous solutions are composed materials which are highly transparent and often are far from spherical. As a result, errors in sizing/counting are inevitable. Any optical technique which is employed for particle analysis relies on differences between the particles refractive index and optical absorption and that of the surrounding medium. When these differences are small, the particle may be wholly or partially undetected. In light obscuration, such particles may either not cause a signal reduction which exceeds the threshold for detection or, may cause a reduction which is smaller than that corresponding to a PS calibration sphere having the same ESD.
Another disadvantage of the light obscuration technique is the limited range of particle concentrations that the technique is capable of handling. In light obscuration, if more than one particle is present in the beam, the signal reductions will be added resulting in errors in size and concentration. This limits the maximum concentration of particles which may be present in a sample to approximately 16 thousand per cc, with existing instruments. For samples with unknown concentration, successive dilutions must be carried out until further dilution does not influence the distributions measured.
Measuring size of particulates using visual microscopy also has disadvantages. Visual microscopy is a manual operation, and, therefore, is prone to a subjectivity, error, and fatigue of an operator. Moreover, preparation of samples for microscopic analysis is a lengthy and costly procedure which can only be done by specially trained personnel.
The apparatus described in the present invention is highly tolerant to concentration and refractive index variations of particles being detected in a fluid. The apparatus does not require calibration by an operator, nor does it require a priori information about particle parameters such as size, shape, or transparency. In fact, these parameters can be measured directly for each particle detected. The end user of present invention has an additional benefit of collecting vast information about particle parameter statistics and selecting particle sub-populations based on those statistics, so as to highlight information about particles of interest. In particular, the addition of information on shape parameters is valuable in assessing patient outcomes. Shape and morphology data are also valuable in assisting to identify particle origin for formulation development, stability assessment, process control, quality control, diagnostics and troubleshooting.
The invention allows one to make quantitative measurements which do not rely on operator judgment thereby eliminating human subjectivity and fatigue as a source of error. The skill level, required to operate the apparatus of present invention, is less than that required to perform microscopic analysis. Besides, the invention can be applied to analyze samples in their native form eliminating the cost and time associated with the preparation of microscopic samples. It can also be used to process larger volumes of parenteral formulations over extended periods of time with no degradation in performance.